Biosci. Biotechnol. Biochem., 77 (8), 1628–1632, 2013

Glycosaminoglycan and Collagen Facilitate the Degradation of Pigment Epithelium-Derived Factor by Chymotrypsin

Akio SAITO

Division of Basic Medicine, Faculty of Medicine, Kinki University, 377-2 Ohno-higashi, Osaka-sayama, Osaka 589-8511, Japan

Received February 12, 2013; Accepted April 22, 2013; Online Publication, August 7, 2013 [doi:10.1271/bbb.130069]

Pigment epithelium-derived factor (PEDF) is a mem- exact mechanisms underlying these diverse activities ber of the serine protease inhibitor family. It is present remain to be resolved, PEDF might prove an effective in a variety of tissues and organs, including plasma. therapeutic agent in treatment.12,13) Here, PEDF was purified from human plasma by use of In the current study, I purified PEDF from human a dermatan sulfate affinity column, and then hydro- plasma by DS affinity column, and investigated its xyapatite, gel filtration and ion exchange columns. It did interaction with proteases in the presence of GAGs and not form a complex with various proteases, including collagen. I hypothesized that GAGs induce a conforma- chymotrypsin, elastase, kallikrein, thrombin, , tional change in PEDF, and that the association of GAGs cathepsins G, activated C, and urokinase, but with PEDF indicates cryptic inhibitory activity against a collagen type I facilitated the degradation of PEDF by specific protease, but PEDF did not form a stable chymotrypsin more than 10-fold. Dermatan sulfate, complex with any of the proteases examined, and GAG heparan sulfate, and heparin showed similar effects, but and collagen had no effect on complex formation. By other glycosaminoglycans, such as hyaluronic acid, contrast, degradation of PEDF by chymotrypsin and by chondroitin sulfate A, C, D, E, and keratan sulfate, elastase was facilitated by DS, heparan sulfate, heparin, had no effect on PEDF degradation. and collagen type I.

Key words: pigment epithelium-derived factor (PEDF); Materials and Methods chymotrypsin; glycosaminoglycan; collagen Materials. Pooled human plasma was purchased from Cosmo Bio Pigment epithelium-derived factor (PEDF) was first (Tokyo). PEG-6000, ammonium sulfate, ammonium chloride, and isolated from retinal pigment epithelium cells as a factor reagents for SDS–PAGE were from Wako Pure Chemical Industries 1,2) (Osaka, Japan). DEAE Sepharose, Superdex 200 10/300 GL, Mono Q, with potent neuronal differentiative activity. Subse- and PD-10 columns were from GE Healthcare UK (Buckinghamshire, quently, and cDNA sequence data revealed UK). A hydroxyapatite column (CHT2-I) was from Bio-Rad Labo- that it belongs to the serine protease inhibitor () ratories (Hercules, CA). Recombinant PEDF was from Peprotech family.3) The protease inhibitory activity of recombinant (Rocky Hill, NJ). Anti-PEDF (rabbit polyclonal IgG) and anti-rabbit PEDF was demonstrated by Becerra et al.,4) who also IgG AP-linked antibodies were from Upstate (Temecula, CA) and Cell found that it was inactive against serine proteases such Signaling Technology (Danvers, MA), respectively. Immobilon P was as trypsin, chymotrypsin, elastase, and cathepsin G. from Millipore (Bedford, MA). Succinyl-phenyalanyl-prolyl-phenyl- alanine p-nitroanilide was from Bachem (Bubendorf, Switzerland). They compared the sequence of the reactive center loop The following proteases were from Sigma (Saint Louis, MO): porcine (RCL) of PEDF with that of other serpin molecules, and trypsin, bovine chymotrypsin porcine pancreas elastase, kallikreins observed that the failure of PEDF to inhibit might be due from human plasma and porcine pancreas, human plasmin, human to unfavorable amino acid residues at P12, P10, and P8, thrombin, and human leukocyte cathepsin G. Human urokinase was even though PEDF shares the P1 residue (Leu) with from ProSpec-Tany TechnoGene (Rehovot, Israel). Human activated inhibitory such as antichymotrypsin and heparin protein C (APC) was from Hematologic Technologies (Essex Junction, cofactor II. VT). Collagen type I (bovine achilles tendon) and polyvinylpyrolidone 5) 40 (PVP-40) was from Sigma. Pfu pyroglutamate aminopeptidase was Alberdi et al. found that PEDF binds to glycosami- from Takara Bio (Kusatsu, Japan). The following GAGs were from noglycans (GAGs) such as heparin, chondroitin sulfate- Seikagaku (Tokyo): hyaluronic acid (pig skin), CSA (sturgeon A (CSA), chondroitin sulfate-C (CSC), dermatan sulfate notochoid), DS (pig skin), CSD (shark cartilage), CSE (squid (DS), and dextran sulfate, and then analyzed the GAG- cartilage), chondroitin (shark cartilage), keratin sulfate (bovine binding region by chemical modification and limited cornea), and heparan sulfate (bovine kidney). Heparin from pig proteolysis. PEDF, also called serpin F-1,6) was origi- intestine was from Wako. were measured with a Pierce BCA protein assay kit (Pierce, Rockford, IL). nally identified in the fetal human , is expressed throughout the human body and is present in blood at a 7) Preparation of PEDF from human plasma. To 30 mL of human concentration of about 100 nM. It exhibits a variety of plasma was added 2.7 g of PEG 6000 (9% saturation) for 1 h on ice. biological functions, including antiangiogenic, antitu- After centrifugation, 3.3 g of PEG 6000 (20% saturation) was added to morigenic, and neurotrophic ones.8–11) Although the the supernatant, and this was mixed for 1 h on ice. The precipitate was

Correspondence: Fax: +81-72-366-0245; E-mail: [email protected] Abbreviations: PEDF, pigment epithelium-derived factor; RCL, reactive center loop; GAG, glycosaminoglycan; CSA, chondroitin sulfate A; DS, dermatan sulfate; TB, Tris–HCl pH 7.5; ECM, extra-cellular matrix Degradation of Pigment Epithelium-Derived Factor by Chymotrypsin 1629 collected by centrifugation, dissolved in 5 mL of 20 mM Tris–HCl mixed with -cyano-4-hydroxy cinnamic acid, and subjected to mass pH 7.5 (TB) containing 0.1 M ammonium chloride, and loaded onto a spectrometry analysis using Voyager STR Biospectrometry Work- DEAE Sepharose column (bed volume 10 mL). The flow-through station (Applied Biosystems Japan, Tokyo). fraction (30 mL) was collected and subjected to ammonium sulfate fractionation. The precipitate that formed between 45% and 80% Amidase activity of chymotrypsin. Chymotrypsin (40 ng) was ammonium sulfate saturation was dissolved in 7 mL of TB, and the dissolved in 500 mL of TBS, and 5 mL of succinyl-phenylalanyl- ammonium sulfate was removed by passage through a PD-10 column. prolyl-phenylalanine-p-nitroanilide (20 mM in N-methyl-2-pyrrolidone) Recovery of protein from the plasma sample at this stage was about was added. The mixture was incubated at 37 C for 30 min, and the 17%. absorbance at 410 nm was measured. To measure the inhibitory effect The ammonium sulfate precipitate (AS45/80) was subjected to of GAGs on chymotrypsin activity against PEDF in solution, PEDF 14) affinity chromatography with DS or heparin (GE Healthcare). An and GAGs were incubated for 10 min prior to the addition of aliquot of AS45/80 (18.7 mg of protein obtained from 2 mL of original chymotrypsin. plasma sample) was applied to a DS column (bed volume 1 mL) equilibrated with TB. The column was washed with 4 mL of TB, followed by 5 mL of 50 mM NaCl in TB, and the bound protein was Results and Discussion eluted with 250 mM NaCl in TB. When a heparin column was used, the bound fraction was eluted with 500 mM NaCl in TB. The approximate Characterization of plasma PEDF yields of bound protein for the DS and heparin columns were 1.3% and PEDF was first identified in the human eye as a 5.4% respectively. protein with a potent neurotrophic function.1) Thereafter, The buffer of the DS or heparin column eluate was changed to 5 mM PBS via a PD-10 column and loaded onto a CHT2-I column (bed it was found to be present in various organs, including 7) volume 2 mL) equipped with AKTA Explore 10S (GE Healthcare). blood plasma. Petersen et al. prepared PEDF from The column was equilibrated with 5 mM PBS at a flow rate of human plasma with a collagen-Sepharose resin. They 1 mL/min. After sample loading, a linear gradient of PBS (5 mM to purified type I collagen from calf skin and conjugated it 400 mM) was applied over 20 min, and 1-mL fractions were collected. to CNBr-activated Sepharose 4B. Next, they applied a An aliquot of the eluent was analyzed by Western blotting with anti- plasma sample to the affinity column and eluted the PEDF. The PEDF fraction from the CHT2-I column was concentrated and applied to a Superdex 200 10/300 GL column equilibrated with bound protein with 500 mM NaCl, thereby achieving a TB containing 0.15 M NaCl (TBS) at a flow rate of 0.5 mL/min. PEDF one-step purification of PEDF. Previously, I attempted was monitored by Western blotting as above, the PEDF fractions were to repeat their purification procedure using commercial combined and concentrated, and the sample buffer of the sample was collagen type I from bovine tendon, but this collagen did then changed to TB. Finally, PEDF was purified by Mono Q column not sufficiently restore plasma PEDF. Having found that with a linear gradient of NaCl in TB from 100 mM to 500 mM over PEDF can bind to a DS affinity column,14) here I applied 18 min at a flow rate of 1 mL/min. a DS column after ammonium sulfate fractionation and then purified PEDF successively by means of hydro- N-Terminal amino acid sequence. Because the N-terminal amino acid was not detected for the intact form of purified PEDF, blocking xyapatite, gel filtration, and ion exchange columns. and removal of the N-terminal amino acid was carried out on the The purified PEDF showed a single band on SDS– membrane. PEDF was subjected to SDS–PAGE, blotted onto a PVDF PAGE and contamination by other proteins was ex- membrane, and stained with CBBR-350 (GE Healthcare). The stained cluded by MALDI-MS analysis upon in-gel digestion of band corresponding to PEDF was cut out and treated with 0.5% PVP- PEDF with trypsin. The single N-terminal sequence,  40 in 100 mM acetic acid (200 mL) at 37 C for 30 min. The PVP-40 Asn-Pro-Ala-Ser-Pro-Pro-Glu-Glu-Gly-, was detected solution was then removed, and the membrane piece was washed with by Edman degradation after pyroglutamate aminopepti- water 10 times. The PEDF on the membrane was treated with pyroglutamate amino peptidase in 10 mLof50mM PB pH 8.0 dase treatment. Without enzyme treatment no amino containing 10 mM DTT, 1 mM EDTA, and 5% glycerol at 37 C for acid was detected, indicating that the N-terminal amino 40 h. Pyroglutamic acid was removed and, the N-terminal sequence of acid of the plasma PEDF was modified by the formation PEDF was analyzed with a gas-phase sequencer (470 Protein of pyroglutamic acid, and that the purified PEDF was Sequencer, Applied Biosystems). not contaminated by other proteins.

Western blotting. Electrophoresis was carried out with a 10% mini- Interaction of PEDF with proteases sized gel (9cm 7:5cm) under reducing conditions for 1 h at 300 volt/30 mA. The gel was overlaid with a PVDF membrane, and Both PEDF and antiplasmin are members of serpin the proteins in the gel were electroblotted by a semi-dry system for clade F, but PEDF is recognized as a non-inhibitory 6) 30 min at 12 volt/120 mA. The membrane was blocked with 3% BSA serpin whereas antiplasmin inhibits trypsin and uroki- for 10 min and treated with anti-PEDF for 3 h. It was then treated with nase as well as plasmin.15) Becerra et al.4,16) demon- secondary antibody conjugated to alkaline phosphatase for 1.5 h and strated interaction of recombinant PEDF with cathepsin visualized with BCIP/NBT (Sigma FAST). G, chymotrypsin, trypsin, thrombin, and elastase but none of these proteases formed a complex with PEDF on Gel chromatography of the complex of PEDF with trypsin. PEDF an SDS–PAGE gel. They also reported that the cleavage (3 mg) was incubated with 0.5 mg of chymotrypsin for 30 min at 37 C, and PMSF was added at 1 mM to prevent excess degradation. Then the site was digested by several proteases, and that mixture was applied to a Superdex 200 10/300 GL column equipped chymotrypsin cleaved Leu-Thr in the RCL region. with AKTA Explore 10S. The column was equilibrated with TBS When trypsin or chymotrypsin was incubated with an at a flow rate of 0.5 mL/min. Thirteen mL was eluted and 0.5 mL- inhibitory serpin such -1-antitrypsin, an SDS-resistant fractions were collected. The fractions (10 mL) were resolved by SDS– complex band was observed on an SDS–PAGE gel. PAGE under reducing conditions, followed by Western blotting with Figure 1 shows the results of the incubation of PEDF anti-PEDF. with various proteases as analyzed by SDS–PAGE under reducing conditions followed by Western blotting. As Analysis of the peptide sequence by mass spectrometry. The proteins were analyzed by SDS–PAGE and stained with silver. The reported previously, cathepsin G, chymotrypsin and protein bands were excised, and the protein was digested with trypsin elastase did not form complexes, but they cleaved PEDF overnight at 37 C in the gel. The digested peptides were extracted, to different extents (lanes 2–4). Other proteases, includ- 1630 A. SAITO

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Fig. 1. Interaction of PEDF with Proteases. PEDF (50 ng) was incubated with various proteases in 10 mLof Fig. 3. Interaction of PEDF with Chymotrypsin and Elastase in the  TB at 37 C for 30 min. The samples were separated by electro- Presence of GAGs and of Collagen. phoresis on a 10% gel and subjected to Western blotting using anti- PEDF (50 ng) was incubated with chymotrypsin (20 ng each, lanes PEDF. Lane 1, control (without protease); lane 2, cathepsin G 2–8) and elastase (20 ng each, lanes 9–15) in the presence of 5 mgof (20 ng); lane 3, chymotrypsin (200 ng, 12.4 munits); lane 4, elastase hyaluronic acid (lanes 3 and 10), chondroitin sulfate E (lanes 4 and (200 ng, 1.88 munits); lane 5, thrombin (200 ng, 0.1 NIH units); 11), DS (lanes 5 and 12), heparin (lanes 6 and 13), keratan sulfate lane 6, urokinase (200 ng); lane 7, plasma kallikrein (200 ng); (lanes 7 and 14), or collagen type I from tendon (lanes 8 and 15) in lane 8, tissue kallikrein (200 ng). Molecular weight standards 10 mL of TB. After incubation for 30 min at 37 C, the samples were (kDa) are indicated on the right. subjected to electrophoresis on a 10% gel, followed by Western blotting using anti-PEDF. Lane 1 is a control for PEDF, and lanes 2 and 9 are controls for chymotrypsin and elastase activities AB respectively. Molecular weight standards (kDa) are indicated on 40 91011121314 M 8 9 10111213 the right. 75

50 second peak (25 kDa) might have been free chymotryp- sin. These data indicate that PEDF associated with 37 30 chymotrypsin but dissociated in the course of SDS– PAGE.

Interaction of PEDF with chymotrypsin and elastase 20 One of the characteristics of the PEDF molecule is that it binds to collagen and hyaluronic acid in addition to sulfated GAGs. Among the serpins, III, heparin cofactor II, and are known to 10 bind to heparin and DS,14) but the ability to bind 17) 2164 6 8 10 12 14 hyaluronic acid is unique to PEDF. Because chymo- trypsin and elastase cleaved PEDF, the effects of glycosaminoglycans and collagen on the interaction of Fig. 2. Gel Filtration Profile of PEDF and the Complex with Chymotrypsin. these two proteases with PEDF was investigated. As PEDF (3 mg) and a mixture of PEDF (3 mg) and chymotrypsin shown in Fig. 3, 20 ng of chymotrypsin (lanes 2–8) or (0.5 mg) were applied to a Superdex 200 column and resolved as elastase (lanes 9–15) was incubated with PEDF in the described in the text. Broken curve and solid curve represent PEDF presence and the absence of GAGs or collagen type I. and the complex respectively. The numbers on the abscissa are Although the amount of protease was not sufficient to fraction numbers. The absorbance at 215 nm (mAU) is plotted on the ordinate. The insert shows the Western blot of the fractions (PEDF cleave PEDF (lanes 2 and 9), the PEDF band of the in A, the complex in B). Molecular weight (kDa) is indicated on samples containing DS, heparin, and collagen disap- the right. peared (lanes 5, 6, 8, 12, 13, and 15). These three molecules increased the susceptibility of PEDF to ing thrombin, urokinase, plasma kallikrein, and tissue degradation by chymotrypsin and elastase. As shown kallikrein, were almost inactive (lanes 5–8). Activated in lane 9, some of the PEDF (49 kDa) was degraded to a protein C and plasmin were also inactive (data not 46-kDa fragment under these reaction conditions, but shown). PEDF is present in plasma at a concentration of the fragment band did not increase with hyaluronic acid, 100 nM,7) but the proteases present in the plasma were keratan sulfate, or chondroitin sulfate E. When recombi- not active against plasma PEDF. Then complex for- nant PEDF expressed in E. coli was used in place of mation was investigated by gel chromatography. PEDF plasma-derived PEDF, chymotrypsin in the presence of was incubated with chymotrypsin and applied to a DS, heparin and collagen degraded recombinant PEDF Superdex 200 column, and the elution of it was in a manner similar to that observed for plasma PEDF monitored by Western blotting. As shown in Fig. 2, (data not shown). Thus the carbohydrate chain in plasma PEDF incubated with chymotrypsin eluted earlier than PEDF was not responsible for the selective degradation intact PEDF. The major peak was fraction 12 for PEDF of PEDF by the proteases. and 10 for the complex. The molecular weight of the complex, indicated by the first peak of the chromato- Effects of GAGs and collagen on the digestion of gram (Fig. 2 solid curve) was estimated to be 67 kDa, PEDF by chymotrypsin but the molecular weight of the complex on Western Because DS and heparin were found to facilitate the blotting was the same as that of intact PEDF, and the degradation of PEDF by chymotrypsin and elastase, Degradation of Pigment Epithelium-Derived Factor by Chymotrypsin 1631 1 243 56789 10 11 1213 14 15 AB 1 2345678 12345678 75

50 75 37 50 25 37 25 20 Fig. 4. Effects of GAGs and Collagen on the Interaction of PEDF with Chymotrypsin. PEDF (50 ng) was incubated with 20 ng of chymotrypsin (lanes 2– 15) in the presence of 5 mg of GAG (lanes 3–13) or collagen (lanes Fig. 5. Acceleration of PEDF Interaction with Chymotrypsin by 14 and 15) in 10 mL of TB for 30 min at 37 C. Lane 3, hyaluronic GAGs and Collagen. acid; lane 4, chondroitin sulfate A; lane 5, DS; lane 6, chondroitin PEDF (400 ng) was incubated with various amounts of chymo- sulfate C; lane 7, chondroitin sulfate D; lane 8, chondroitin sulfate trypsin in the absence (lanes 2–4) and the presence (lanes 5–8) of E; lane 9, chondroitin; lane 10, keratan sulfate; lane 11, heparan GAG or collagens in 10 mL of TB for 30 min at 37 C. One-tenth of sulfate; lane 12, heparin; lane 13, dextran sulfate; lane 14, collagen each sample was removed and subjected to Western blotting after type I (tendon); lane 15, collagen type IV (placenta). Then the electrophoresis on a 12% gel. The rest of each sample was resolved samples were subjected to electrophoresis on a 10% gel and Western on a 12% gel, and the gel was stained with silver. Lane 1, PEDF blotting with anti-PEDF. Molecular weight standards (kDa) are only; lane 2, 20 ng of chymotrypsin; lane 3, 75 ng of chymotrypsin; indicated on the right. lane 4, 200 ng of chymotrypsin; lanes 5–8, 20 ng of chymotrypsin and 10 mg of hyaluronic acid (lane 5), 10 mg of DS (lane 6), 5 mgof collagen type I (lane 7), or 5 mg of collagen type IV (lane 8). A, other chondroitin sulfates and related molecules, Western blotting; B, silver staining. Molecular weight standards together with collagen type IV, were investigated. As (kDa) are indicated on the right. shown in Fig. 4, heparan sulfate and dextran sulfate, in addition to DS and heparin, were found to be effective. PEDF might, in one sense, protect against attack on With respect to collagen, type I from tendon tissue PEDF by the protease; however, the RCL region of the accelerated the cleavage of PEDF, but type IV from serpin molecule is situated away from the GAG and the placenta was ineffective. Because PEDF is known to collagen binding sites. Thus, it is likely that chymo- bind to collagen types I and III but not to collagen types trypsin attacks the RCL of PEDF, resulting in a II and IV,18) type IV collagen would not be expected to conformational change in PEDF. This indicates that influence the interaction of PEDF with chymotrypsin. the binding profile of DS and that of heparin are The acceleration of PEDF degradation by chymo- different from that of other GAGs such as CSA, CSC, or trypsin was investigated in more detail. As shown in hyaluronic acid.21) On the other hand, Valnickova et al. Fig. 5, lane 4, the PEDF band was detected on a silver- investigated conformational changes in PEDF by limited stained gel and a Western blot after incubation of 400 ng proteolysis with trypsin, and found that hyaluronic acid, of PEDF with 200 ng of chymotrypsin. Becerra et al. chondroitin sulfate, and collagen were resistant to investigated the digestion of PEDF by chymotrypsin proteolysis while heparin modified the proteolytic sus- with a much lower protease-substrate ratio w/w ratio of ceptibility and PEDF was cleaved.22) DS and heparan 1:100,16) but they used PEDF isolated from bovine eyes. sulfate, but not CSA, also activate plasma kallikrein, Plasma PEDF may be more resistant to digestion by which is responsible for the cleavage of the factor H proteases (1:2 w/w ratio). When 10 mgofDSor5mgof molecule in the plasma.23) DS, heparan sulfate, and collagen type I was included in the incubation, however, collagen are abundant in the extracellular matrix (ECM), PEDF was completely degraded by 20 ng of chymo- and the diverse biological activity of PEDF, including trypsin (lanes 6 and 7). Alberdi et al.5) observed that antiangiogenic and antitumorigenic activities, must be PEDF bound to Sepharose columns conjugated with affected by these ECM molecules. CSA and CSC, in addition to DS, and that the bound In conclusion, PEDF was purified from human PEDF was eluted with 100–150 mM NaCl. It is possible plasma, and the effects of GAGs and collagen on the that DS, but not CSA or CSC, induces a conformation in cleavage of PEDF by chymotrypsin was investigated. PEDF that makes it susceptible to digestion by chymo- DS, heparan sulfate, heparin, and dextran sulfate trypsin. Another possibility is that DS stimulates the facilitated the degradation of PEDF by chymotrypsin, activity of chymotrypsin. When chymotrypsin activity but other GAGs such as hyaluronic acid, CSA, or CAC was measured in solution using a chromogenic substrate were not effective. In addition, collagen type I but not in the presence and the absence of DS, no significant type IV facilitated PEDF degradation. increase in activity was observed (data not shown). Similarly, other GAGs and collagen did not affect the References activity of chymotrypsin in solution. Thus the acceler- ation of PEDF degradation was due to the fact that DS 1) Tombran-Tink J and Johnson LV, Invest. Ophthalmol. Vis. Sci., bound to PEDF not to the enzyme. 30, 1700–1707 (1989). The detailed structure of PEDF indicates that a basic 2) Tombran-Tink J, Chader GG, and Johnson LV, Exp. 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